The interaction of rabbit skeletal muscle troponin-T fragments with troponin-I

Abstract

The interactions of troponin-I (Tn-I) with a variety of fragments spanning the length of the troponin-T (Tn-T) polypeptide chain have been reinvestigated at physiological ionic strength by affinity chromatographic, gel filtration, and circular dichroism methodologies. Strong binding was observed with fragment T2 (residues 159-259) mimicking that observed with whole Tn-T and Tn-I. Partial binding was seen with the shorter cyanogen bromide (CB) fragments of Tn-T in the order CB4 (residues 176-230) greater than CB6 (residues 239-259) or CB5 (residues 152-175). No interaction with Tn-I was observed with fragments (CB2, CB3, T1) encompassing residues 1-158 of Tn-T. Based on the present results and the work of others, the binding region for Tn-I includes residues 159-259 and perhaps extends into the highly helical CB2 region (residues 71-151) of Tn-T. No evidence has been obtained by ourselves or others for the interaction of the CB3 region (1-70) with Tn-I. A significant increase (11.6%) in alpha-helical content was observed when an equimolar amount of fragment T2 (residues 159-259) was mixed with Tn-I, a result similar to that seen with whole Tn-T and Tn-I.

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

“…Two other α-helices of cTnI are observed in this structure, cTnI and cTnI . While cTnI is free of contact with cTnC and cTnT, cTnI 90-136 forms a coiled-coil with a portion of the T2 fragment of cTnT, cTnT , as predicted previously [38]. Upstream from the coiled-coil, cTnT 204-220 forms another α-helix ( Figure 1A).…”

Investigation of the effects of phosphorylation of rabbit striated muscle αα‐tropomyosin and rabbit skeletal muscle troponin‐T

“…Also unresolved is the matter of the strengthened interaction between the C-terminal portion of Tn-T and phosphorylated TM at binding site 11. Nevertheless, the proposal deserves consideration because, in the presence of Ca"', the attachment of Tn-T to TM at site I represents the main thin filament anchor for Tn (Pearlstone and Smillie, 1983). Assuming this to be true for thin filaments containing phosphorylated TM, can the increase in ATPase activity (Figs 6C and 7) be rationalised in terms of a change in the ability of Tn-T to bridge the overlap site?…”

“…Thus, differences in interaction inferred from polymerisation measurements are predicted not to hold once the components become fixed to actin. A further problem is the fact that relatively little functional significance can be attributed to the outermost part of Tn-T, encompassing the first 45-69 amino acids (Pearlstone and Smillie, 1982;Tobacman, 1988;Pan et al, 1991;Hill et al, 1992;Willadsen et al, 1992), even though this region clearly bridges the overlap (White et al, 1987). Of greater relevance, at least in the assay systems that have been used, is the inner portion of the N-terminal domain, covering residues 70-159 (Willadsen et al, 1992).…”

“…Research into the arrangement of components within the complex has shown Tn-T to cover a considerable length of the TM molecule and to interact at two positions Pearlstone and Smillie 1983). One of these is situated in the locality of Cysl90 of TM (site 11), and the other at the overlap (site I).…”

“…These alterations are a product not only of distinct stmctural genes but also exon splicing. First, apparent from the amino acid sequence comparison of three chicken isoforms (Wilkinson et al, 1984), splicing has been observed in both skeletal and cardiac Tn-Ts (Hastings et al, 1985;Cooper and Ordahl, 1985;Risnik et al, 1985;Breibart and Nadal-Ginard, 1986;Pearlstone et al, 1986;Leszyk et al, 1987;Gahlmann et al, 1987;Smillie et al, 1988;Briggs and Schachat, 1989;Fujita et al, 1991;Jin et al, 1992). While not all of the splicing possibilities are realised at the protein level (Briggs et al, 1990), heterogeneity at the N-terminal end has been linked to changes in the Ca2+ sensitivity of reconstituted thin filaments (Tobacman and Lee, 1987;Tobacman 1988).…”